Survival In Ewing Sarcoma Research Articles

High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability

A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

Referral patterns, treatment and outcome of high-grade malignant bone sarcoma in Scandinavia--SSG Central Register 25 years' experience.

The objectives of this study were to present changes in referral patterns, treatment and survival in patients with high-grade malignant bone sarcoma in Sweden and Norway based on data in the Scandinavian Sarcoma Group (SSG) Central Register. Data on 1,437 patients with diagnosis 1986-2010 was analyzed. Osteosarcoma was the most frequentl diagnosis (45%), followed by Ewing sarcoma (21%) and chondrosarcoma (17%). Thirty-one percent of Swedish and 41% of Norwegian patients had tumors in the axial skeleton. Eighty-six percent of extremity tumors and 66% of axial tumors were referred to a sarcoma center prior to unplanned surgery or biopsy. During the past decade, limb salvage surgery has risen from under 50% to over 80%. Five-year overall survival in non-metastatic osteosarcoma was 70% for extremity tumors, and 35% for axial tumors. No improvement in osteosarcoma survival was observed during the last decade. Five-year survival in Ewing sarcoma improved from 50% to 69%. Referral patterns in bone sarcomas have improved. However, greater efforts should be dedicated to improving referral of patients with possible tumors in the axial skeleton to multidisciplinary teams (MDTs). Overall survival of patients with high-grade malignant bone sarcomas in Sweden and Norway is in line with other reports.

MicroRNA and Bone Cancer.

MicroRNA molecules have a variety of roles in cellular development and proliferation processes, including normal osteogenesis. These effects are exerted through post-translational inhibition of target genes. Altered miRNA expression has been demonstrated in several cancers, both in the tumor tissue and in the peripheral circulation. This may influence carcinogenesis if the specific miRNA targets are encoded by tumor suppressor genes or oncogenes. To date, most research investigating the role of microRNAs and primary bone tumors has focused on osteosarcoma and Ewing sarcoma. Several microRNAs including the miR-34 family have been implicated in osteosarcoma tumorigenesis via effects on the Notch signaling pathway. Progression, invasion, and metastasis of osteosarcoma tumor cells is also influenced by microRNA expression. In addition, microRNA expression may affect the response to chemotherapy in osteosarcoma and thus hold potential for future use as either a prognostic indicator or a therapeutic target. The EWS-FLI1 fusion protein produced in Ewing sarcoma has been shown to induce changes in miRNA expression. MicroRNA expression profiling may have some potential for prediction of disease progression and survival in Ewing sarcoma. There is limited evidence to support a role for microRNAs in other primary bone tumors, either malignant or benign; however, early work is suggestive of involvement in chondrosarcoma, multiple osteochondromatosis, and giant cell tumors of bone.

Abstract 3422: RNAi screening identifies FGFR4 as a modulator of growth and survival in Ewing sarcoma

Abstract Ewing sarcoma is the second most common cancer of bone and soft tissue arising in children and young adults. Standard treatment of Ewing sarcoma includes surgery, radiation, and chemotherapy largely consisting of combinations of non-targeted cytotoxic agents. Although the survival rate has improved for patients treated for localized disease, the survival rate of patients with metastatic tumor remains lower than 30%. In order to improve therapeutic options for Ewing Sarcoma, we employed a functional genomics approach based on RNA interference (RNAi) screening to identify genes whose silencing affected the proliferation and survival of Ewing sarcoma cells. Four Ewing sarcoma cell lines, TC-32, TC-71, SK-ES-1 and RD-ES, were transfected with a library of siRNA targeting 287 cancer-associated genes. The siRNA screening data were normalized and statistical cut-offs were determined for each cell line. From the list of siRNA ‘hits,’ we identified siRNAs targeting Fibroblast growth factor 4, FGFR4, to be among the most effective in inducing cytotoxicity in Ewing sarcoma cells. Further validation was conducted showing that siRNAs to FGFR4, but not FGFR1, FGFR2, or FGFR3, were able to significantly reduce Ewing Sarcoma cell viability. Furthermore, siRNA targeting FGFR4 were able to induce Caspase 3 activity. FGFR4 protein was shown to be expressed in Ewing sarcoma by western blot analysis, with expression levels lower than FGFR4 expression on rhabdomyosarcoma cells. Targeting FGFR4 activity in Ewing sarcoma cells using a pan-FGFR inhibitor, PD-173074, demonstrated that Ewing sarcoma cells were sensitive to FGFR inhibition. Since FGFR4 in a potential therapeutic target in rhadomyosarcoma, we compared the response of the Ewing sarcoma cells to PD-173074 to that of the rhabdomyosarcoma cell lines, RH-30 and RD. The IC50s of the four Ewing sarcoma cells ranged from 3.8-4.7 µM and were slightly lower than the IC50 for the rhabdomyosarcoma cell lines RH-30 (5.6 µM) and RD (7.9 µM). These results indicate that FGFR4 is a potential therapeutic target in Ewing sarcoma and that FGFR4 targeted therapy should be tested in preclinical xenograft models of Ewing sarcoma. Citation Format: David O. Azorsa, Irma M. Gonzales, Shilpi Arora, R. Tanner Hagelstrom, Tanya H. Little, Robert J. Arceci, Spyro Mousses. RNAi screening identifies FGFR4 as a modulator of growth and survival in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3422. doi:10.1158/1538-7445.AM2014-3422

Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma

Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of > 20% in ES tumors (range 24–71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of > 20% (range 36–50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types.

Long-term Survivors of Childhood Ewing Sarcoma: Report From the Childhood Cancer Survivor Study

The survival of Ewing sarcoma (ES) patients has improved since the 1970s but is associated with considerable future health risks. The study population consisted of long-term (> or =5-year) survivors of childhood ES diagnosed before age 21 from 1970 to 1986. Cause-specific mortality was evaluated in eligible survivors (n = 568), and subsequent malignant neoplasms, chronic health conditions, infertility, and health status were evaluated in the subset participating in the Childhood Cancer Survivor Study (n = 403). Outcomes were compared with the US population and sibling control subjects (n = 3899). Logistic, Poisson, or Cox proportional hazards models, with adjustments for sex, age, race/ethnicity, and potential intrafamily correlation, were used. Statistical tests were two-sided. Cumulative mortality of ES survivors was 25.0% (95% confidence interval [CI] = 21.1 to 28.9) 25 years after diagnosis. The all-cause standardized mortality ratio was 13.3 (95% CI = 11.2 to 15.8) overall, 23.1 (95% CI = 17.6 to 29.7) for women, and 10.0 (95% CI = 7.9 to 12.5) for men. The nonrecurrence-progression non-external cause standardized mortality ratio (subsequent non-ES malignant neoplasms and cardiac and pulmonary causes potentially attributable to ES treatment) was 8.7 (95% CI = 6.2 to 12.0). Twenty-five years after ES diagnosis, cumulative incidence of subsequent malignant neoplasms, excluding nonmelanoma skin cancers, was 9.0% (95% CI = 5.8 to 12.2). Compared with siblings, survivors had an increased risk of severe, life-threatening, or disabling chronic health conditions (relative risk = 6.0, 95% CI = 4.1 to 9.0). Survivors had lower fertility rates (women: P = .005; men: P < .001) and higher rates of moderate to extreme adverse health status (P < .001). Long-term survivors of childhood ES exhibit excess mortality and morbidity.